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. 2017 May;69(5):1111-1121.
doi: 10.1002/art.39982. Epub 2017 Mar 31.

CRISPR/Cas9 Editing of Murine Induced Pluripotent Stem Cells for Engineering Inflammation-Resistant Tissues

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Free PMC article

CRISPR/Cas9 Editing of Murine Induced Pluripotent Stem Cells for Engineering Inflammation-Resistant Tissues

Jonathan M Brunger et al. Arthritis Rheumatol. .
Free PMC article

Abstract

Objective: Proinflammatory cytokines such as interleukin-1 (IL-1) are found in elevated levels in diseased or injured tissues and promote rapid tissue degradation while preventing stem cell differentiation. This study was undertaken to engineer inflammation-resistant murine induced pluripotent stem cells (iPSCs) through deletion of the IL-1 signaling pathway and to demonstrate the utility of these cells for engineering replacements for diseased or damaged tissues.

Methods: Targeted deletion of the IL-1 receptor type I (IL-1RI) gene in murine iPSCs was achieved using the RNA-guided, site-specific clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 genome engineering system. Clonal cell populations with homozygous and heterozygous deletions were isolated, and loss of receptor expression and cytokine signaling was confirmed by flow cytometry and transcriptional reporter assays, respectively. Cartilage was engineered from edited iPSCs and tested for its ability to resist IL-1-mediated degradation in gene expression, histologic, and biomechanical assays after a 3-day treatment with 1 ng/ml of IL-1α.

Results: Three of 41 clones isolated possessed the IL-1RI+/- genotype. Four clones possessed the IL-1RI-/- genotype, and flow cytometry confirmed loss of IL-1RI on the surface of these cells, which led to an absence of NF-κB transcription activation after IL-1α treatment. Cartilage engineered from homozygous null clones was resistant to cytokine-mediated tissue degradation. In contrast, cartilage derived from wild-type and heterozygous clones exhibited significant degradative responses, highlighting the need for complete IL-1 blockade.

Conclusion: This work demonstrates proof-of-concept of the ability to engineer custom-designed stem cells that are immune to proinflammatory cytokines (i.e., IL-1) as a potential cell source for cartilage tissue engineering.

Figures

Fig 1
Fig 1
Schematic illustrating our strategy to generate iPSCs resistant to IL-1-mediated signaling for tissue engineering applications. (A) Binding of IL-1 ligand to the IL1R1 receptor results in activation of a pro-inflammatory transcriptional program involving the transcription factors NF-κB, JNK, and MSK1. (B) Guide RNAs (gRNAs) target the genome editing nuclease Cas9 to two sites flanking exon 2 of IL1R1, which encodes the signal peptide sequence. (C) Cas9 induces DNA double-strand breaks (DSBs), which may be repaired via a DNA repair pathway known as non-homologous end joining. (D) This will lead to a subset of alleles with fully intact IL1R1, while others may have genomic disruptions at the IL1R1 locus, including excision of the signal peptide sequence, resulting in loss of signaling through IL1R1.
Fig 2
Fig 2
(A) Genomic PCR from clones isolated after single-cell deposition. PCR amplicons represent the presence or absence of exon 2 in the Il1r1 locus. (B) Sanger sequencing from an allele with the CRISPR/Cas9-mediated deletion of exon2 from Il1r1. (C) Flow cytometry histograms demonstrating differential levels of Il1r1 surface expression in populations derived from each of the Il1r1+/+, Il1r1+/- and Il1r1-/- genotypes. (D) Luminescence data characterizing the transcriptional activity of NF-κB in Il1r1+/+, Il1r1+/-, and Il1r1-/- cells after a 24 hour treatment with 1 ng/ml IL-1α. Bars represent group means ± SEM (n=4). Groups not sharing the same letter are statistically different (p<0.05).
Fig 3
Fig 3
Relative gene expression data as measured by qRT-PCR to examine the effects of IL-1α treatment on engineered cartilage derived from either Il1r1+/+, Il1r1+/-, or Il1r1-/- cells. Bars represent group means of fold change ± SEM (n=4). Groups not sharing the same letter are statistically different (p<0.05).
Fig. 4
Fig. 4
(A, B) Biochemical analyses of engineered cartilage composition. (A) Double-stranded DNA (dsDNA) content and (B) sulfated glycosaminoglycan (sGAG) per DNA as measured by the picogreen and dimethylmethylene blue (DMMB) assays, respectively. Bars represent group means ± SEM (n=6). *p<0.05 between Il1r1+/- and other genotypes. Groups not sharing the same letter are statistically different (p<0.05). (C) Representative images from Safranin-O/fast green/hematoxylin staining of 10 μm sections of engineered cartilage treated with or without 1 ng/ml of IL-1 for 72 hours. Scale bar = 500 μm.
Fig. 5
Fig. 5
Analysis of media samples collected from engineered cartilage aggregates cultured with or without 1 ng/ml IL-1α for 72 hours. (A) Specific MMP activity (n=7). RFU indicates relative fluorescence units. (B) Concentration of sGAG measured in culture media (n=6). (C) PGE2 concentration (n=4). (D) Total nitric oxide concentrations (n=4). Bars represent group means ± SEM. Groups not sharing the same letter are statistically different (p<0.05).

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